Kit for detection/quantification of analyte, and method for detection/quantification of analyte

Abstract

Disclosed are a detection/quantification kit and a detection/quantification method for detecting/quantifying an analyte rapidly at a low cost and in a simple manner. Specifically, disclosed is a kit for detecting an analyte in a sample, comprising a first conjugate and a second conjugate, wherein the first conjugate comprises a first substance comprising a stimuli-responsive polymer and a first affinity substance having affinity for the analyte and bound to the first substance, and the second conjugate comprises a second substance carrying an electrical charge and a second affinity substance having affinity for the analyte and bound to the second substance. The first and second affinity substances can bind to different sites on the analyte simultaneously.

The invention claimed is: 
     
       1. A method for detecting a target substance in a sample, comprising steps of:
 mixing a first bound substance in which a first substance containing stimuli-responsive polymer binds to a first affinity substance having affinity to the target substance, a second bound substance in which the second bound substance is electrically charged binds to a second affinity substance having affinity to the target substance, and a sample; wherein said the second bound substance inhibits the aggregation of stimuli responsive polymer; 
 putting the mixture under aggregation conditions of the stimuli-responsive polymer, and determining if the stimuli-responsive polymer is dispersed or not, wherein 
 the first affinity substance and the second affinity substance can simultaneously bind to different sites of the target substance; and determining that the target substance is present in a case in which aggregation inhibition of the stimuli-responsive polymer is confirmed via inhibition of the aggregation of the stimuli responsive polymer by the second bound substance, and that the target substance is not present in a case in which aggregation inhibition of the stimuli-responsive polymer is not confirmed; wherein the step of determining if dispersion is present or not is carried out under aggregation conditions of the stimuli-responsive polymer. 
 
     
     
       2. The method according to  claim 1 , wherein the first substance comprises a particulate magnetic material, and the method further comprises a step of separating the aggregated magnetic material by applying a magnetic force. 
     
     
       3. A method for quantifying a target substance in a sample, comprising steps of:
 mixing a first bound substance in which a first substance containing stimuli-responsive polymer binds to a first affinity substance having affinity to the target substance, a second bound substance in which the second bound substance is electrically charged binds to a second affinity substance having affinity to the target substance, and a sample; wherein said the second bound substance inhibits the aggregation of stimuli responsive polymer; 
 putting the mixture under aggregation condition of the stimuli-responsive polymer, and measuring a change of the turbidity of the mixture via inhibition of the aggregation of the stimuli responsive polymer by the second bound substance; and 
 calculating the amount of a target substance in the sample based on a correlation equation between the amount of the target substance and the turbidity under the aggregation conditions wherein the step of measuring the turbidity is carried out under aggregation conditions of the stimuli-responsive polymer. 
 
     
     
       4. The method according to  claim 3 , wherein the first substance comprises a particulate magnetic material, and the method further comprises a step of separating the aggregated magnetic material by applying a magnetic force. 
     
     
       5. A kit for detecting a target substance, comprising: a first bound substance, wherein a first substance including a stimuli-responsive polymer binds to a first affinity substance having affinity to the target substance; and a second bound substance in which a second electrically charged substance binds to a second affinity substance having affinity to the target substance, wherein the first affinity substance and the second affinity substance can bind simultaneously to different sites of the target substance, wherein said kit detects said target substance according to a method for detecting a target substance in a sample, comprising steps of: mixing a first bound substance in which a first substance containing stimuli-responsive polymer binds to a first affinity substance having affinity to the target substance, a second bound substance in which the second bound substance is electrically charged binds to a second affinity substance having affinity to the target substance, and a sample; wherein said the second bound substance inhibits the aggregation of stimuli-responsive polymer; putting the mixture under aggregation conditions of the stimuli-responsive polymer, and determining if the stimuli-responsive polymer is dispersed or not, wherein the first affinity substance and the second affinity substance can simultaneously bind to different sites of the target substance; and determining that the target substance is present in a case in which aggregation inhibition of the stimuli-responsive polymer is confirmed via inhibition of the aggregation of the stimuli-responsive polymer by the second bound substance, and the target substance is not present in a case in which aggregation inhibition of the stimuli-responsive polymer is not confirmed; wherein the step of determining if dispersion is present or not is carried out under aggregation conditions of the stimuli-responsive polymer. 
     
     
       6. The kit according to  claim 5 , wherein the first substance comprises a particulate magnetic material. 
     
     
       7. The kit according to  claim 6 , wherein the second substance is a hydrophilic polymer compound. 
     
     
       8. The kit according to  claim 7 , wherein the second substance is a polyanion or a polycation. 
     
     
       9. The kit according to  claim 8 , wherein the polyanion is a nucleic acid or a polyacrylic acid. 
     
     
       10. The kit according to  claim 8 , wherein the polycation is a polyalkylamine or a polyethyleneimine. 
     
     
       11. The kit according to  claim 5 , wherein the second substance is a hydrophilic polymer compound. 
     
     
       12. The kit according to  claim 11 , wherein the second substance is a polyanion or a polycation. 
     
     
       13. The kit according to  claim 12 , wherein the polyanion is a nucleic acid or a polyacrylic acid. 
     
     
       14. The kit according to  claim 12 , wherein the polycation is a polyalkylamine or a polyethyleneimine.